1. Field of the Invention
The present invention relates to a process for preparing a polyacrylamide gel plate for use in electrophoresis, and, more particularly, to a process for preparing a large quantity of polyacrylamide gel plates which are especially suitable for electrophoresis analysis of in vivo components with a high molecular weight, such as proteins and the like.
2. Description of the Background Art
Polyacrylamide gels have widely been used for electrophoresis analysis of in vivo components with a high molecular weight, e.g., protein, nucleic acid, and the like. Usually, these polyacrylamide gels are prepared by cross-linking polymerization of a monomer and a cross-linking agent; i.e., by adding a polymerization initiator to a 2-40% by weight aqueous solution comprising a monomer such as acrylamides and a divalent or polyvalent cross-linking agent such as N,N'-methylenebisacrylamide (such a solution is hereinafter sometimes referred to as an "acrylamide monomer solution").
As an initiation method of polymerization, either a combination of a peroxide and a reducing agent, or a combination of a peroxide, a reducing agent, a photosensitation agent and exciting ray, while the reducing agent is not necessarily used, may be used. For the latter photopolymerization method, although some methods are proposed, in which the polymerization does not take place before the light irradiation, even after a polymerization initiator added (e.g., Japanese Patent Laid-open (kokai) No. 91849/1987), the photopolymerization method basically involves many technical problems for obtaining stable gel products with a good reproducibility. The problems are found in difficulties in controlling the polymerization reaction such as shading of the reaction liquid for gel formation, selection of irradiation conditions for producing gel, and occurrence of continued polymerization in the presence of light even after the completion of gel formation. For these reasons, the former chemical polymerization method, which adopts a combination of a peroxide and a reducing agent, is preferred owing to its comparatively easy handling of the reaction liquid and capability of controlling the reaction to some extent by adjusting the amount of the catalyst used.
In the chemical polymerization method, however, since the gel reaction takes place immediately after the addition of the polymerization initiator to the monomer solution, giving rise to a viscosity increase of the solution and to gelatinization of the mixture, it is necessary to prepare a monomer solution and a polymerization initiator solution separately and mix them just before the start of the gel forming process. This separate addition of a peroxide solution and a reducing solution to the monomer solution inevitably requires complicated steps. On the other hand, if these two solutions are mixed together beforehand, the catalyst activity will change as the time passes, making it difficult to stably obtain high quality gels.
Furthermore, since a molecular sieve effect of a polyacrylamide gel varies depending on the gel concentrations, various products with different gel concentrations are required conforming to molecular weights or the like of the components to be separated or analyzed. Conventionally, this requirement for polyacrylamide gels has been fulfilled by preparing a monomer solution having a specific gel concentration, which corresponds to the subject to be separated for analysis, then adding a polymerization initiator to the solution and introducing the mixture into or spreading it over a gel-supporter. This necessitates to prepare many gel-forming solutions with different concentrations when the subjects to be analyzed contain a number of components. In addition, preparation of a gel with a concentration gradient suitable for the analysis of a subject comprising a wide molecular weight distribution is implemented by providing two monomer solutions with different concentrations, one a low concentration and the other a high concentration, and adding a polymerization initiator to the solutions. The solutions are then introduced into or spread over a gel-supporter by using a gradient forming unit. In order to obtain a number of gels with different concentration gradients, it is necessary, in the same manner as in the preparation of a series of gels with a specified concentration, to prepare beforehand various monomer solutions with different concentrations corresponding to the required gradients, and to appropriately combine these solutions. Furthermore, in order to prepare a stable gel, the amount of the polymerization initiator must be adjusted appropriately so that not too less or not too much of it is incorporated relative to the concentration of the monomer solution to be used.
As noted above, stable supply of high quality polyacrylamide gels in large quantities has been difficult to achieve by conventional preparation methods which involve rather complicated manufacturing steps and require sophisticated skills.
An object of the present invention is, therefore, to provide an easy production process which can prepare a large quantity of various kinds of high quality and stable acqueous polyacrylamide gels, having a high resolving power and any desired concentrations or concentration gradients, with a good reproducibility, and which does not involve the problem of changes in the polymerization initiator activity before being introduced into a gel-supporter or does not require to prepare many monomer solutions or many polymerization initiator solutions of different concentrations beforehand.
In view of this situation, the present inventors have undertaken intensive studies to solve the aforementioned problems, and found that a high quality gel with a desired concentration can constantly and easily be prepared with a good reproducibility and without any changes in the catalyst activity using an optimum amount of a polymerization initiator, by simply preparing one monomer solution with a high concentration, one peroxide solution with a low concentration, and one reducing solution with a low concentration, and mixing them at an appropriate ratio prior to use. These findings have led to the completion of the present invention.